BIOSCAN QC Report

Statistics for the positive controls

Total number of positive controls: 96 
Number of positive controls per plate: 1 

All plates have positive controls: TRUE  
Total number of reads in positive controls: 29562 
Maximum number of reads: 466 in positive control sample: CONTROL_POS_BAYS_017_G12 
Minimum number of reads: 3 in CONTROL_POS_FACE_201_G12 

Average number of positive control reads: 307.9375 
Median number of positive control reads: 317 
Read standard deviation: 85.9480641832154 

Quantiles:
 5%: 128.5
10%: 191.5
25%: 279
50%: 317
75%: 354.25
95%: 436
100%: 466

Blue solid line: read mean
Orange dotted lines: 5% and 10% lower quantiles

Number of positive control samples in the lower 5% quantile: 5 

 CONTROL_POS_FACE_201_G12
CONTROL_POS_FACE_202_G12
CONTROL_POS_FACE_205_G12
CONTROL_POS_FACE_207_G12
CONTROL_POS_FACE_208_G12 

Names of the associated partners: BIFOR

Statistics for the negative controls

Total number of negative controls: 621 
Total number of lysate negative controls: 525 
Total number of empty negative controls: 96 

Number of negative controls per plate:

Number of negative controls per plate	Number of plates
2	87
more than 2	9


All plates have negative controls: TRUE 

Total number of reads in lysate negative controls: 1629 
Total number of reads in empty negative controls: 86 
Maximum number of reads: 292 in lysate negative control sample: CONTROL_NEG_LYSATE_FACE_203_H12 
Maximum number of reads: 5 in empty negative control sample: CONTROL_NEG_BAYS_019_A2 

Zero reads in: 424 negative control samples 
In lysate controls: 381 
In empty controls: 43 

Average number of negative control reads: 2.76167471819646 
In lysate controls: 3.10285714285714 
In empty controls: 0.895833333333333 

Median number of negative control reads: 0 
In lysate controls: 0 
In empty controls: 1 

Skewness number of negative control reads: 11.0469717733312 
In lysate controls: 10.1363579769267 
In empty controls: 1.30385483117931 

Quantiles in lysate controls:
 5%: 0
10%: 0
25%: 0
50%: 0
75%: 1
95%: 3
98%: 15.5599999999999 

Quantiles in empty controls:
 5%: 0
10%: 0
25%: 0
50%: 1
75%: 1
95%: 3
98%: 3.09999999999999

Blue solid line: read mean
Orange dotted lines: upper 5% and 2% of samples with the highers number of reads

Number of negative control samples in the higher 5%: 39 
 Out of in the lysate controls: 30 
 Out of in the empty controls: 9 
 
Number of negative control samples in the higher 2%: 13 
 Out of in the lysate controls: 13 
 Out of in the empty controls: 0 

Names of the associated partners: BAYS, CAMP, BIFOR, LFLA, RRHP, WTPV

Statistics for the samples

Number of samples in the batch (exclusing controls): 8499 
Total number of partner plates: 96 
Total number of sample reads: 2832708 

Maximum number of sample reads: 906 in sample: LFLA_010_A2 
Minimum number of sample reads: 0 in 310 samples
 which is 3.64748793975762 % of all samples 

Average number of reads: 333.298976350159 
Median number of reads: 360 
Read standard deviation: 200.8572183961 
Skewness number of sample reads: -0.170122229845947 

Quantiles:
 5%: 1
10%: 18
25%: 166.5
50%: 360
75%: 489
95%: 636
100%: 906

Blue solid line: read mean
Orange dotted lines: lower 5% and 10% of samples

Number of samples in the lower 10%: 851 out of 8499 samples 
Number of samples in the lower 5%: 461 out of 8499 samples 

Partners associated with the bottom 5% of samples by read count:

Partner names	Frequency
RRHP	176
BIFOR	127
LFLA	70
BAYS	57
WTPV	23
CAMP	8


Number of samples with 0 reads: 310

Plate boxplots

Plates where the 75th percentile of the data is lower than expected mean read count (dark grey):

 FACE_006
FACE_010
FACE_011
FACE_013
FACE_017
FACE_020
FACE_201
FACE_202
FACE_203
FACE_204
FACE_205
FACE_206
FACE_207
FACE_208
RRHP_026
RRHP_027
RRHP_061
WTPV_014 
 
which constitutes 18.75 % of all partner plates in this batch

Grey line: median
Brown line: mean
Green data points: positive controls
Blue data points: empty negative controls
Navy data points: lysate negative controls

Plates where the 75th percentile of the data is lower than expected mean read count (dark grey): 4, 7, 8 
How many samples from the low-performance partner plates are present in the low-performance UMI plates (purple data points): 55.9230306674684 %

Assess the positive controls with the low number of reads detected in the previous steps:

FACE_201 Positive control failed.
 Observed number of reads: 3 Expected: 102.161290322581 

FACE_202 Positive control failed.
 Observed number of reads: 55 Expected: 61.8817204301075 

FACE_205 More reads in positive control than in samples on average.
 Observed number of reads: 127 Expected: 50.4086021505376 

FACE_207 More reads in positive control than in samples on average.
 Observed number of reads: 119 Expected: 66.5698924731183 

FACE_208 Positive control failed.
 Observed number of reads: 75 Expected: 118.075268817204

FACE_205 
FACE_207 
FACE_208 
FACE_201 
FACE_202

The above plates have lower than expected number of reads 
AND failed positive controls. 
THESE PLATES NEED TO BE EXAMINED FURTHER

Low-quality plates are displayed here. All the other plates are plotted in the last part of this report.
Green squares: controls [any kind]

Assessment of sequence conflicts and contaminants

Positive control as contamination source

NOTE: All sample and sequence IDs match - data successfully merged

Positive control OTU is TAX:1287025 

Non-positve control samples that contain positive control reads:

Sample	Control Sequence Count	Sequence Similarity	Sequence Type	UMI Plate ID
BAYS_002_F11	1	99.84779	secondary	9
BAYS_015_B6	1	99.84825	secondary	13
BAYS_019_E8	1	99.84871	secondary	14
LFLA_003_G8	1	99.84848	secondary	19
LFLA_012_A5	1	98.31547	secondary	15
RRHP_026_A4	1	99.84802	secondary	4
RRHP_027_D8	1	100.00000	primary	5
WTPV_020_H2	1	99.69697	secondary	17

Number of samples with positive control OTU as primary sequence: 1 
Number of samples with positive control OTU as secondary sequence: 7 
out of 5838 samples with secondary sequences 

Location of the contaminants relative to the source:

Orange square: positive contros
Green squares: samples with positive control contamination

Read count mean of all secondary sequences in all samples: 4.96586979401594 
Read count mean of all positive control sequences in other samples: 1 

Read count median of all secondary sequences in all samples: 1 
Read count median of all positive control sequences in other samples: 1

Blue solid line: secondary hit read mean
Orange dotted lines: mean of reads found as secondary contaminants from the positive controls in other samples
Both lines should be in close proximity meaning that the secondary contamination from positive controls is comparable to the potential contamination in other samples.

NOTE: Non-control samples with control reads recognised as the primary hit need to be examined further!

Sample	Count	OTU	Sequence
RRHP_027_D8	1	TAX:1287025	primary

NOTE: the above samples are automatically removed if:

There’s only one primary read

Secondary sequence found in the same sample is not an Arthropod

Negative control contamination

Distribution of reads in negative controls

NOTE: contamination source can be either primary or secondary sequence within samples!

Family	No. Source Samples
Chironomidae	73
Tachinidae	51
Hominidae	9
Culicidae	7
Anthomyiidae	5
Dolichopodidae	3
Platygastridae	2
Agromyzidae	1
Aleyrodidae	1
Aphididae	1
Cecidomyiidae	1
None	1
Scelionidae	1
Tipulidae	1

Outline: negative controls with contaminants
Colour of the oultine indicates partners to track the samples between partner and UMI plates.
Thicker chartreuse outline: FAILED negative controls with contaminants [2%]
Numbers indicate the read count
Squares that are not outlined represent potential sources of contamination within plates: identical sequences found within these wells and negative controls.

Assessment of primary and secondary sequences

NOTE: Controls are not included! 

Number of wells with a primary sequence only: 2327 
Number of wells with primary and secondary sequences: 5794 

Number of primary chimeric sequences: 56 
Number of secondary chimeric sequences: 7600 

NOTE: All secondary chimeric sequences successfully removed

[1] 6
Number of samples with only primary chimeric sequence recognised: 28 
We do not know how mBRAVE recognises chimeras - for now ony samples represented by less than 5 reads get removed
Retained samples: 6

Number of EXCLUDED primary sequences: 499 
 which constitutes 6.16125447586122 % of all samples 
These samples are not being removed - it's an mBRAVE cut-off 

Number of primary sequences with no taxonomy assigned: 608 
 which constitutes 7.5070996419311 % of all samples 
These samples are going to be examined further

Number of samples with no taxonomy assigned that will be replaced with the secondary sequence based on the sequence similarity: 150 
Other sequences with no taxonomy assigned to the primary sequence will remain unchanged.

If the first entry is not ‘Arthropod’, then the second entry is likely correct [based on manual observations]

Number of samples with Wolbachia detected: 335 

Table with plate positions, number of reads, and sequences saved to the output directory:
 /nfs/users/nfs_a/aw43/aw43/2024_07_bioscan_qc/input/output/qc_reports/batch18/

Number of samples with Nematoda, Tardigrada, Annelida, and/or Rotifera detected: 80 

Table with plate positions, number of reads, and sequences saved to the output directory:
 /nfs/users/nfs_a/aw43/aw43/2024_07_bioscan_qc/input/output/qc_reports/batch18/

Taxon	Frequency
Chordata	20
Nematoda	9
Proteobacteria	54
Rotifera	2

85 wells had primary non-Arthropod hits and secondary Arthropod hits 
NOTE: Primary hits are going to be replaced
 
Samples with only non-Arthropod sequences detected: 29 
NOTE: These samples have been excluded!

Number of African Anopheline samples: 50 
Number of primary African Anopheline hits [250 or more reads]: 24 
NOTE: All primary mosquito samples removed!

Number of samples with only primary Arthropod sequence: 5779 
 73.1982267257758 % of all remaining samples 

Number of samples where secondary sequence is not present elsewhere on the partner or UMI plate: 0 
Number of conflicting sequences [sequences are in different families or orders, both have good read support]: 231

Primary hit	Number
Arthropoda	7210
None	531

Number of retained samples: 7741 
Number of Arthropod samples assigned by mBRAVE [this inscludes samples with 5 or less reads that have now been excluded!]: 7376 
Number of samples with replaced sequences: 34 
Retained chimeras: 20 
Retained samples with no taxonomy: 531 

Each retreived sample has only one sequence: TRUE

Number of samples	Description	Category	Decision
4402	Only one sequence with more than 200 reads, no secondary sequence detected	1	YES
863	Only one sequence with 50 to 200 reads, no secondary sequence detected	2	YES
482	Only one sequence with 5 or more but less than 50 reads, no secondary sequence detected	3	YES
123	Dominant sequence with more than 200 reads, non-conflicting secondary sequences with 5 or less reads	4	YES
48	Dominant sequence with 50 to 200 reads, non-conflicting secondary sequences with 5 or less reads	5	YES
671	Dominant sequence with more than 200 reads, conflicting secondary sequences with 5 or less reads	6	YES
376	Dominant sequence with 50 to 200 reads, conflicting secondary sequences with 5 or less read	7	YES
279	Dominant sequence with more than 200 reads, secondary sequences with more than 5 read support	8	NO
177	Dominant sequence with 50 to 200 reads, secondary sequences with more than 5 read support	9	NO
251	Dominant sequence with 5 or more but less than 50 reads, non-conflicting secondary sequences with less than 5 reads	10	NO
69	Dominant sequence with more than 5 but less than 50 reads, any other secondary reads present	11	NO

Decision category	Number of samples
NO	776
YES	6965

8.91869631721379 % OF SAMPLES EXCLUDED [all samples]

18.0491822567361 % OF SAMPLES EXCLUDED [only approved samples]

Plate heatmaps - retained samples

NOTE: The heatmaps below show only the retained samples. Controls, chimeric samples, non-Arthropod samples, and samples with no taxonomy assigned have been removed or replaced!

Final fasta file succesfully saved: /nfs/users/nfs_a/aw43/aw43/2024_07_bioscan_qc/input/output/qc_reports/batch18/BOLD_filtered_sequences_batch18.fasta 
Final metadata file succesfully saved: /nfs/users/nfs_a/aw43/aw43/2024_07_bioscan_qc/input/output/qc_reports/batch18/BOLDfiltered_metadata_batch18.csv

The report and output files have been successfully generated!

Number of retained samples per partner plate

Plate	Original number of samples	Number of samples after filtering	Percentage
WTPV_018	93	93	100.000000
RRHP_063	14	14	100.000000
WTPV_025	49	48	97.959184
BAYS_018	93	91	97.849462
FACE_011	93	91	97.849462
RRHP_045	93	91	97.849462
WTPV_016	93	91	97.849462
WTPV_019	93	91	97.849462
RRHP_061	93	90	96.774193
FACE_009	93	89	95.698925
FACE_010	93	89	95.698925
LFLA_010	93	89	95.698925
WTPV_020	93	89	95.698925
BAYS_022	93	88	94.623656
WTPV_013	93	88	94.623656
WTPV_015	93	88	94.623656
LFLA_014	18	17	94.444444
BAYS_003	93	87	93.548387
WTPV_014	93	87	93.548387
WTPV_022	93	87	93.548387
LFLA_002	31	29	93.548387
CAMP_025	75	70	93.333333
BAYS_014	93	86	92.473118
CAMP_036	93	86	92.473118
RRHP_029	93	86	92.473118
RRHP_048	53	49	92.452830
FACE_006	93	85	91.397850
WTPV_021	93	85	91.397850
WTPV_024	93	85	91.397850
CAMP_029	93	84	90.322581
CAMP_034	93	84	90.322581
CAMP_035	93	84	90.322581
FACE_012	93	84	90.322581
FACE_014	93	84	90.322581
LFLA_001	93	84	90.322581
RRHP_046	93	84	90.322581
RRHP_062	93	84	90.322581
BAYS_002	93	83	89.247312
BAYS_015	93	83	89.247312
BAYS_019	93	83	89.247312
CAMP_031	93	83	89.247312
FACE_007	93	83	89.247312
FACE_008	93	83	89.247312
FACE_013	93	83	89.247312
RRHP_047	93	83	89.247312
WTPV_017	93	83	89.247312
BAYS_020	93	82	88.172043
FACE_213	93	82	88.172043
BAYS_001	93	81	87.096774
CAMP_030	93	81	87.096774
BAYS_011	93	80	86.021505
CAMP_033	93	80	86.021505
LFLA_003	93	80	86.021505
CAMP_024	74	63	85.135135
BAYS_008	93	79	84.946237
WTPV_023	93	79	84.946237
BAYS_017	93	78	83.870968
LFLA_007	93	78	83.870968
BAYS_010	93	77	82.795699
CAMP_032	93	77	82.795699
CAMP_037	93	77	82.795699
FACE_018	93	77	82.795699
LFLA_008	93	77	82.795699
LFLA_004	93	76	81.720430
BAYS_005	93	75	80.645161
FACE_017	93	75	80.645161
BAYS_004	93	74	79.569892
BAYS_016	93	74	79.569892
FACE_016	93	74	79.569892
FACE_210	93	74	79.569892
LFLA_050	93	74	79.569892
FACE_015	93	73	78.494624
BAYS_013	93	72	77.419355
LFLA_005	93	72	77.419355
LFLA_006	93	72	77.419355
BAYS_009	93	71	76.344086
BAYS_012	93	70	75.268817
FACE_212	93	69	74.193548
BAYS_006	93	68	73.118280
LFLA_009	93	66	70.967742
FACE_208	93	64	68.817204
FACE_020	93	63	67.741935
BAYS_007	93	62	66.666667
FACE_217	93	62	66.666667
FACE_211	93	61	65.591398
FACE_204	93	58	62.365591
FACE_202	93	56	60.215054
FACE_205	93	55	59.139785
FACE_203	93	54	58.064516
FACE_207	93	48	51.612903
FACE_201	93	47	50.537634
FACE_218	12	6	50.000000
LFLA_012	93	45	48.387097
FACE_206	93	44	47.311828
RRHP_026	93	3	3.225807
RRHP_027	82	2	2.439024

Number of retained samples per partner

Partner	Original number of samples	Number of samples after filtering	Percentage
WTPV	1165	1094	93.90558
CAMP	986	869	88.13387
BAYS	1953	1644	84.17819
LFLA	1072	859	80.13060
FACE	2523	1913	75.82243
RRHP	800	586	73.25000

Number of retained samples per UMI plate

Plate	Original number of samples	Number of samples after filtering	Percentage
17	372	356	95.69892
16	372	354	95.16129
2	372	353	94.89247
6	332	306	92.16867
14	372	337	90.59140
18	372	336	90.32258
24	372	331	88.97849
1	353	314	88.95184
13	372	326	87.63441
23	372	321	86.29032
3	372	314	84.40860
9	354	298	84.18079
19	328	276	84.14634
12	372	308	82.79570
10	372	304	81.72043
22	291	234	80.41237
20	372	293	78.76344
21	372	293	78.76344
11	372	289	77.68817
5	361	263	72.85319
15	235	165	70.21277
4	372	218	58.60215
7	293	171	58.36177
8	372	205	55.10753

The plates with low number of reads and retained samples should be examined!

Samples to examine manually

Failed negative controls [2%] with contamination other than Bovidae:

 CONTROL_NEG_LYSATE_BAYS_005_H12
CONTROL_NEG_LYSATE_BAYS_007_H12
CONTROL_NEG_LYSATE_BAYS_008_H12
CONTROL_NEG_LYSATE_BAYS_010_H12
CONTROL_NEG_LYSATE_CAMP_025_B1
CONTROL_NEG_LYSATE_CAMP_025_B2
CONTROL_NEG_LYSATE_FACE_203_H12
CONTROL_NEG_LYSATE_FACE_218_A8
CONTROL_NEG_LYSATE_FACE_218_B7
CONTROL_NEG_LYSATE_FACE_218_E8
CONTROL_NEG_LYSATE_FACE_218_F8
CONTROL_NEG_LYSATE_RRHP_027_F11
CONTROL_NEG_LYSATE_WTPV_017_H12

These samples may have insects in them!